Structure-function studies of proteins impose two major requirements on biophysical spectroscopic methods. First, to succeed in such studies, a spectroscopic method should be capable of providing atomic-scale structural data with minimal perturbation to the system. Second, the protein should be maintained in a functionally relevant conformational state. Ideally, one would like to observe detailed molecular structure while the protein progresses through conformational states associated with normal function.
Although modern high-resolution NMR spectroscopy offers unique opportunities for structure-function studies of many water-soluble proteins, membrane proteins are generally less amendable to solution NMR methods. Molecular tumbling of proteins in lipid bilayer systems is insufficient to average out anisotropic magnetic interactions, thus causing broad NMR lines that lead to inevitable loss of resolution and sensitivity. One way to approach the line width problem is to solubilize membrane proteins in mixed detergent micelles that form a shell around the hydrophobic portion of the protein. These detergent-protein complexes may tumble rapidly enough in solution to produce narrow, motionally averaged NMR resonances.
Unfortunately, surrounding some proteins with detergent molecules is known to destabilize functional conformational states and/or perturb the membrane protein tertiary structure (1). There is also growing evidence that many membrane proteins adopt functionally active conformational states only when embedded in or interacting with lipid bilayers of specific composition representative of the native environment (2).
Alternatively, membrane proteins can be studied by solid-state NMR with spectral resolution improved through macroscopic alignment of the surrounding lipid bilayers with respect to the magnetic field axis. Such alignment results in all proteins in the sample having the same polar angle with respect to the bilayer normal perpendicular to the field, therefore decreasing anisotropic line shape contributions. However, longitudinal orientation of the protein in this spherical coordinate system defined by the bilayer normal remains undetermined, and the anisotropy resulting from observations over the longitudinal angle can still lead to line broadening. This remaining anisotropy can be averaged by rapid rotational diffusion of the protein about the lipid bilayer normal (3). Thus, the latter condition constitutes another essential requirement for achieving the best spectral resolution for macroscopically aligned membrane proteins in which nuclear chemical shift tensors are not uniformly aligned with B0.
Generally, lipid bilayer samples are aligned either mechanically by assembling bilayers on planar solid surfaces such as glass (4) or magnetically by forces that arise in external magnetic field for discoidal bilayered micelles (also called bicelles) with sufficient degree of anisotropic magnetic susceptibility (5,6).
Bicelles can be prepared by mixing long-chain phospholipids such as DMPC (1,2-dimyristoyl-sn-glycero-3-phosphocholine) with a short-chain DHPC (1,2-dihexanoyl-snglycero-3-phosphocholine) in a molar ratio greater than approximately 2.5. In these bicelles, DMPC molecules form near planar bilayers that are capped by short-chain DHPC lipids. The negative sign of the bicelles' magnetic susceptibility tensor forces them to align with the bilayer surface parallel to the direction of the magnetic field. Addition of a few mol percent (with respect to the lipids) of certain lanthanide ions flips the bicelle orientation by 90° (7). For solid-state NMR experiments with membrane proteins, the bicelle magnetic field alignment method offers several important advantages including ease of sample preparation, full hydration of lipid bilayers, and long (>1 year) shelf life of biologically stable samples (8). Although several other lipid compositions for bicelles have been reported in the literature, including mixtures that maintain alignment at both acidic and basic pH values (9) and extended temperature range (10), the choice of lipid composition for anisotropic bicelles that would undergo magnetic field alignment remains limited in comparison with the mechanical (or substrate) alignment method. The latter can be successfully employed for bilayers composed from various types of lipids and under an extended range of external conditions such as temperature, ionic strength, pH, etc. (4, 11-13). Therefore, the mechanical alignment method is generally more applicable and is practiced more often in studies of membrane proteins (14,15).
One significant drawback of the substrate-alignment method derives from the somewhat limited ability to modify the sample hydration, pH, ionic strength, and concentration of molecules in the aqueous phase. Consequently, it is virtually impossible to expose the same sample, without lengthy sample regeneration and realignment, to a variety of water-soluble agents that could be used to trigger changes in membrane protein conformation or function. These limitations of the planar substrate-aligned lipid bilayer samples prevent many applications of high-resolution solid-state NMR methods for detailed structure-function studies of membrane proteins.
Nanopore-supported lipid nanotube arrays introduced recently (16) provide an attractive way for aligning membrane protein samples for solid-state NMR experiments (17, 18). In brief, it was shown that these macroscopically aligned nano-tubular bilayers are formed by lipid self-assembly inside nano-porous anodic aluminum oxide (AAO) substrates. FIG. 1 shows a cartoon of a single lipid bilayer that is spreading along a nanopore. AAO substrates have aligned through-film nanoporous channels with exceptionally high density. Examination of typical commercial AAO membranes from Whatman International (Maidstone, UK) with scanning electron microscopy (SEM) have shown that the pore density is at least 109 pores/cm2 with an average pore diameter of 177±20 nm (Alaouie, unpublished results). Thus, for 60 μm thick substrate, the total surface area of the nanoporous channels is 334±38 cm2 for each 1×1×0.006 cm3 AAO strip. Elemental chemical analysis has shown that on average up to four lipid nanotubular bilayers can be deposited into each of these AAO nanopores (19). Then such a deposition would provide approximately the same lipid bilayer surface area as depositing 1300 lipid bilayers on top of a 1 cm2 glass slide. Consequently, by stacking a few tens of AAO strips, one can achieve similar lipid volumes in NMR experiments as in the conventional glass plate method. This was indeed confirmed directly by NMR experiments that demonstrated similar signal/noise ratio for AAO-supported and glass-aligned transmembrane domain of the M2 protein of influenza A virus (17).
Recent studies have shown that the new AAO-supported bilayers retain many biophysical properties of unsupported lipid vesicles (19,20), and they are suitable for aligning membrane proteins for high-resolution multidimensional solid-state NMR studies (17). Detailed NMR (17,18,21,22) and DSC (19,20) studies provided further characterization of the biophysical properties of such nanotubular bilayers. Specifically, 31P NMR studies of POPC (1-palmitoyl-2-oleoyl-snglycero-3 phosphocholine) bilayers absorbed into AAO nanopores indicated that some portions of the bilayer surface were inaccessible to Pr3+ shift reagent when the bilayer was maintained above the main phase transition temperature. Thus, it was concluded that under the latter conditions the ends of the bilayer tubules must be well sealed against the pore such that the ions cannot penetrate into the water underneath the bilayers (21).
Here, we have reexamined the nanotubular bilayer surface accessibility using Mn2+ ions as paramagnetic broadening agents. We demonstrate that, under typical conditions of sample preparation (using DMPC lipids instead of POPC lipids) and handling above and below the main phase transition temperature, both leaflets of nanotubular bilayers are fully accessible to this divalent ion without compromising the macroscopic lipid alignment. Moreover, we demonstrate that the broadening of the 31P resonances is reversible by a gentle treatment of the AAO stack with the chelating agent EDTA (ethylenediaminetetraacetic acid) followed by a straightforward buffer exchange at temperatures above the main phase transition. This indicates that lipid nano-tube arrays can be prepared with the bilayer surfaces fully accessible to water-soluble molecules. Thus, the high hydration levels of these structures as well as pH and desirable ion and/or drug concentrations can be easily maintained and modified. We are particularly interested in exploring these unique features that permit solvent flow through lipid nano-tube arrays for structure-function studies of membrane proteins by solid-state NMR spectroscopy. Here, we present a demonstration of such experiments at high magnetic field (19.6 T) using 17O NMR anisotropic chemical shift effects of ion binding to the gramicidin A channel.
The molecular mechanism of ion selectivity in K+ channels is based on the polypeptide backbone carbonyls (23,24) that provide solvation for K+ on binding. The same fundamental ion-binding interactions are known to contribute to the ion conductance mechanism of other ion transporters including the dimeric gramicidin A (gA) pore, which functions as a monovalent cation-selective channel (25). Gramicidin A is a 15-residue polypeptide with an alternating sequence of D- and L-amino acids:    HCO-L-Val1-Gly2-L-Ala3-D-Leu4-L-Ala5-D-Val6-L-Val7-D-Val8-L-Trp9-D-Leu10-L-Trp11-D-Leu12-L-Trp13-D-Leu14-L-Trp15-NHCH2CH2OH (25). In bilayers formed from lipids with fatty acyl chain lengths matching the hydrophobic dimension of the right-handed b-helix, this polypeptide forms a dimer with a 4.5 Å pore accommodating ions and a single file of water molecules (26,27). Recent NMR studies carried out with gA uniformly aligned in DMPC bilayers using the glass plate method concluded that the carbonyl oxygen of D-Leu10 is one of the three carbonyls involved in the ion-binding site at each end of the channel (28,29).
Specifically, significant perturbation of 17O anisotropic chemical shift was observed when the gA binding sites were predominantly occupied with two potassium cations (29,30). Moreover, it was determined that binding of other ions also affects the 17O NMR chemical shift and that this parameter is a significantly more sensitive indicator for binding events than the 15N chemical shift typically employed in such studies (29) (E. Y. Chekmenev, L. N. Miller, P. L. Gor'kov, Z. Gan, and T. A. Cross, unpublished results). qq
Here we exploit the sensitivity of the 17O chemical shift to the local ion environment for characterizing cation solvation by the Leu10 carbonyl oxygen that helps to form the binding site for ions in the gA pore. In order to macroscopically align the gA-containing bilayers for NMR experiments and to maintain the gA pore fully hydrated and accessible to various ions, we employed lipid nanotube arrays formed inside nanoporous substrates. With the surfaces of both lipid bilayer leaflets fully accessible to aqueous solutes, we were able to utilize physically the same gA sample in a lipid bilayer environment to study reversible effects of mono- and divalent ion binding on the chemical shift properties of gA Leu10 carbonyl oxygen. We also compare the 17O shifts induced by ion binding to those induced by the binding of protons in the pH range from approximately 1 to 12 and find a significant difference. This unexpected result points to a difference in mechanisms for metal ion (28) and proton (31) conduction by the gA pore. Additionally, temperature and pH ranges for the nanopore alignment method are being established. The effect of lipid bilayer phase on gA Leu10 carbonyl 17O line width also has been investigated.